The overall aim of this project is to identify extracellular signals and specific genes involved in the maintenance of hematopoietic stem cells (HSC). An important practical goal of these studies is the establishment of conditions that generate HSC in vitro. Major obstacles to progress in this area have been the inability to purify biologically defined subpopulations of HSC, coupled with the difficulties of identifying the expression (or suppression) of genes involved in HSC self-replication, especially given the very small numbers of HSC available for study (typically a few thousand cells of each defined HSC subpopulation). However, our group has recently developed a flow cytometric method that utilizes a sequential Hoechst 33342/Rhodamine 123 approach to purify distinct subpopulations of HSC suitable for investigations at the cellular and molecular levels. Specifically, we plan: a) to define the specific growth conditions [e.g., growth factors, growth inhibitors, adhesion molecules/ligands ("adhesins")] that control the maintenance of "sternness" of long-term repopulating Hoechst 33342/Rhodamine 123-selected HSC in vitro using single cell cultures and analysis of the proliferative potential of HSC daughter cells by recloning in vitro or assaying in vivo using suitable long-term or short-term repopulation assays; b) to define unique genes expressed (or suppressed) by freshly isolated Hoechst 33342/Rhodamine 123-selected HSC subpopulations that can be physically separated and are biologically distinct (based on the short-term versus long-term repopulating ability in vivo of two subpopulations) using a novel polymerase chain reaction-based subtractive cloning method; c) to define unique genes expressed (or suppressed) in vitro by descendant daughter cells generated from long-term repopulating Hoechst 33342/Rhodamine 123-selected HSC exposed to specific growth factors, + /- proliferation inhibitors and/or adhesins.